Scientific publications described in this application are incorporated in full by reference thereto.
Following their release and action on receptors, monoamine neurotransmitters are taken up from the synaptic cleft into pre-synaptic terminals by plasma membrane transporters. This re-uptake terminates the action of the neurotransmitter. Uptake mechanisms for radiolabeled biogenic amines were first demonstrated by Axelrod and co-workers for noradrenaline Axelrod, J. and Hertting, G., Nature, 192, 172 (1961); Axelrod, J., Science 173, 589 (1971)), and subsequently, for serotonin by several groups (Axelrod, J. and Inscoe, J. K., J. Pharmacol. Exp. Ther. 141, 161 (1963); Aghajanian G. K. and Bloom, F. E., J. Pharmacol. Exp. Ther. 156, 23 (1967); Blackburn, K. J. et al., Life Sci. 6, 1653 (1967)).
In addition to its role in removing serotonin (5-HT) from the synaptic cleft, the 5-HT transporter (5HTT) allows platelets and rodent mast cells to concentrate 5-HT (Pletscher, A., Int. J. Cardiol. 14, 177 (1987)). These cells store and secrete large amounts of the amine, but do not synthesize it.
5-HT transporters are a site of action for some anti-depressants and drugs of abuse such as amphetamines and cocaine.
A cDNA library was constructed from rat basophilic leukemia cell (RBL 2H3, a cognate mast cell) mRNA in pCDM7 (Kanner, B. I. and Bendahan, A., Biochim. Biophys. Acta, 816, 403 (1985) and pools of recombinants were screened by expression of recombinant plasmid-encoded proteins in COS cells. The pCDM7 vector is the same as pCDM8 (Seed, B., Nature 329, 840 (1987)) but without the polyoma origin and a BamHI site. In this vector, cDNAs can be expressed from either the T7 RNA polymerase or the cytomegalovirus virus (CM) promoter. Expression from the T7 polymerase promoter can be accomplished either by transfection into cells expressing T7 polymerase or by in vitro transcription from the T7 promoter ("Protocols and Applications Guide", c. 1991 by Promega Corporation) and microinjection of the synthetic mRNA into a recipient cell.
Poly(A) enriched RNA was prepared from RBL 2H3 cells (cognate mast cell) using guanidinium isothiocyanate followed by oligo(dT) cellulose chromatography (Okayama, H. et al, Methods in Enzymology (Academic Press, Inc., New York) 154, 3 (1987)). Double-stranded cDNA was synthesized from 5 ug RBL 2H3 poly(A)+RNA using Murine moloney reverse transcriptase (Superscript, BRL) and Avian myoblastosis virus reverse transcriptase H.C. (Promega) by the method of U. Gubler and B. J. Hoffman (Gene 25, 263 (1983)). BstXI adaptors (Invitrogen) were ligated to blunt-ended cDNA and size-fractionated on a potassium acetate gradient. cDNA (&gt;1.5 kb) was ligated into BstXI-digested pCDM7 and electroporated into Escherichia coli MC1061p3 to yield a library of 2.3.times.10.sup.6 recombinants.
Plasmid DNA from twenty-four pools of 13,000 recombinants was prepared by Triton X-100 lysis and cesium chloride banding (Chen, C. and Okayama, H., Mol. Cell Biol. 7, 2745 (1982)). DNA (3.3 ug) from each subdivision was transfected onto 2.times.10.sup.5 COS-7 cells in single chamber slides (Lab-Tek) by calcium phosphate precipitation (ibid). After 72 h, cells were washed with uptake buffer consisting of 25 mM Hepes, pH 7.4, 125 mM NaCl, 4.8 mM KCl, 1.2 mM KH.sub.2 PO.sub.4, 1.3 mM CaCl.sub.2, 1.2 mM MgSO.sub.4, 5.6 mM glucose, 1 mM Na ascorbate and 10 uM pargyline, then pre-incubated in uptake buffer for 15 min. Cells were incubated for 2 h with .sup.3 H-5-HT (100 nM) at 37.degree. C. followed by three 1 ml washes on ice, fixed in 2.5% glutaraldehyde in PBS with acrolein (1:100) for 30 min at 23.degree. C. and washed in phosphate-buffered saline (PBS). Following an H.sub.2 O dip to remove salts, slides were air-dried, coated with nuclear emulsion (NTB2, Kodak) and exposed for 2 days. A single positive pool was identified microscopically and further subdivided. At the level of 100 clones per pool, subdivisions were screened using the recombinant T7 RNA polymerase-containing vaccinia virus (Fuerst, T. R. et al., Proc. Natl. Acad. Sci. USA 83, 8122 (1986); Fuerst, T. R. et al., Mol. Cell Biol. 7, 2538 (1987)). 2.times.10.sup.5 CV-1 cells were plated in 6 well plates, infected with T7 RNA polymerase-containing vaccinia virus at a multiplicity of infection of 10 pfu per cell. After 30 min, cells were transfected with 3 ug plasmid DNA isolated from pooled clones of the pCDM7 cDNA transformants using 10 ug lipofectin (BRL). After 24-30 h., cells were assayed for serotonin uptake as described above. Following washes, cells were solubilized in 0.5N NaOH and radioactivity determined by liquid scintillation counting.
In parallel with the functional assay of cDNA clones, subdivisions were screened with a degenerate oligonucleotide: EQU 5'-taggggatca ggaaggcgcc gccnccrtty ttnymrcaca ggtagggga ccgccacaca tt-3'
(SEQ. ID. NO. 3) directed at a region highly conserved in noradrenaline (Pacholczyk, T. et al., Nature 350, 350 (1991)) and GABA transporters (Guastella, J. et al., Science 249, 1303 (1990), Nelson, H. et al., FEBS Lett. 269, 181 (1990)). A single hybridizing band was present in each positive pool identified by bioassay through three successive rounds of screening. Screening with the kinased consensus oligonucleotide probe was performed in 6.times. SSC and 2.times. Denhardt's solution at 60.degree. C. A single positive clone was identified from a positive pool of 100 clones using this consensus oligonucleotide.
B. Sequence Analysis at 5HTT encoding cDNA
5HTT cDNA was subcloned into M13 bacteriophage and sequenced completely on both strands using the Sequenase kit (US Biochemicals). Sequence analysis was performed with the University of Wisconsin Genetics Computer Group Sequence Analysis Package, version 7.0 (1991).
Sequence analysis of the 3.0 kilobase (kb) insert revealed an open reading frame of 1,959 base pairs (bp) (FIG. 2) (SEQ. ID. NO. 1), predicting a protein of 653 amino acids with a relative molecular mass of .about.73,000 (73 kD) excluding glycosylation. The initiating ATG is 52 bases downstream from a stop codon, and the surrounding sequence conforms to a consensus translation initiation site (Kozak, M., Nucleic Acids Res. 15, 8125 (1987)). Hydropathy analysis (Kyte, J.and Doolittle, R. F., J. Mol. Biol. 157, 105 (1982)) indicates 12-13 potential transmembrane domains with no apparent signal sequence (von Heijne, G., Eur. J. Biochem. 133, 17 (1983)). On this basis and by analogy to GABA and noradrenaline transporters (GAT-1 and NET, respectively), the amino- and carboxy-termini, which have potential protein kinase C phosphorylation sites, may be located intracellularly; a large loop with two potential glycosylation sites would then be found extracellularly.